The overarching theme of this project is to explore the mechanisms that lead to immune regulation of alcoholic liver disease (ALD). We will focus on extracellular vesicles (EVs) of gut and hepatic macrophages. Specifically, we will examine how microbial antigen stimulates the production of gut EVs through TLR4 signaling and the consequences of these events in the development of ALD via gut-liver axis. Novel in vitro and in vivo approaches will be used to address mechanisms of action with the goal of identifying new targets for intervention. We propose that gut-derived EVs regulate the accumulation and polarization of hepatic macrophages during alcohol-induced chronic inflammation via TLR4 signaling. We specifically propose that TLR4-activated extracelluar miRNAs production is the key to the activation of macrophages. Based on our published work and other preliminary data, this proposal will test the hypothesis that microbial antigen regulates the production of gut extracellular miRNAs through TLR4 signaling, which initiates a crosstalk among macrophages and hepatocytes in ALD. Moreover, we will determine whether diet-associated nanoparticles carrying inflammatory miRNAs inhibitor have potential therapeutic implications in ALD. These hypotheses will be tested in the following Specific Aims: Aim 1: Determine the role of TLR4-regulated gut EVs on alcohol-induced liver inflammation and injury. Aim 2: Determine whether alteration of IEC miRNAs via TLR4 modulation affects the severity of alcohol-induced liver inflammation and injury in ALD. Following the successful completion of the above Specific Aims, our novel studies will (i) validate TLR4 activated EVs is directly relevant to the activation of hepatic macrophages in ALD, (ii) define the preventive potential of TLR4-extracellular miRNAs axis in a mouse model of ALD and (iii) provide a foundation for future mechanistic investigation of gut EVs and extracellular miRNA in ALD therapy.